We propose to continue our analyses of the E3 transcription unit of adenovirus (Ad) in order to gain insights into how a common pre-mRNA precursor is processed into different mRNAs depending on the selection of alternative splice sites and/or polyadenylation sites. E3 is expressed during "early" stages of infection as 4 major overlapping mRNAs. Our approach has been to construct viruses with mutations throughout E3, then determine how the mutations affect the accumulation of E3 mRNAs in Ad- infected cells. The mutants are viable and the E3 mRNAs are expressed abundantly in a "natural" regions (I and II) where mutations have dramatic effects on the synthesis of E3 mRNAs, and two other regions (III and IV) where mutations have less dramatic effects. we propose that regions I and II, and perhaps regions III and IV, may be discrete elements that act as determinants of alternative pre-mRNA processing. Much of the application deals with the molecular mechanism by which these putative elements function. Two of the E3 mRNAs are scarce at early stages of infection but are expressed abundantly at late stages via splicing to RNA leaders coded far upstream of E3. A major goal is to elucidate why there is early-stage and late-stage-specific use of certain splice and polyadenylation sites. This study should provide insights into the stage-specific regulation of alternative pre-mRNA processing. We will combine genetic and biochemical approaches, including the establishment of in vitro RNA processing systems, identification of cis-acting RNA secondary structures, and attempts to identify trans-acting factors that bind to the pre-mRNA.